Coronary heart disease (CHD) remains the leading cause of death in the industrialized countries. The primary cause of CHD is atherosclerosis, a disease characterized by the deposition of lipids, including cholesterol, in the arterial vessel wall, resulting in a narrowing of the vessel passages and ultimately hardening the vascular system.
It is now well accepted that atherosclerosis can begin with local injury to the arterial endothelium followed by proliferation of arterial smooth muscle cells from the medial layer to the intimal layer along the deposition of lipid and accumulation of foam cells in the lesion. As the atherosclerotic plaque develops it progressively occludes more and more of the affected blood vessel and can eventually lead to ischaemia or infarction. Since deposition of circulating lipids such as cholesterol plays a major role in the initiation and progression of atherosclerosis, it is important to identify methods and compositions to lower circulating cholesterol levels.
Circulating lipoproteins serve as vehicles for the transport of insoluble lipids like cholesterol esters, triglycerides and the more polar phospholipids and unesterified cholesterol in the aqueous environment of plasma (Bradely, W. A. and Gotto, A. M.: American Physiological Society, Bethesda, Md., (1978) pp 117-137). The solubility of these lipids is achieved through physical association with proteins termed as apolipoproteins and the lipid-protein complexes are called lipoproteins (Dolphin, P. J.: Can. J. Biochem. Cell. Biol. (1985) 63, 850-869). Five distinct classes of lipoproteins, chylomicrons, very low density lipoproteins (VLDL), low density lipoproteins (LDL), high density lipoproteins (HDL) and LP(a), have been isolated from human plasma (Alaupovic, P.: In Handbook of Electrophoresis. (1980) Vol. 1, pp. 27-46, Havel, R. J., Eder, H. A. and Bragdon, J. H.: J. Clin. Invest. (1955) 34, 1343-1353). LDL is the major carrier of cholesterol in human plasma.
Dietary triglycerides and cholesterol are assembled by enterocytes (intestinal cells) into a chylomicron particle, which enters circulation through the lymphatic system (Brown, M. S. and Goldstein, J. L Sci. American (1984) 251, 58-66). Chylomicrons provide fatty acids to peripheral cells and cholesterol to liver. The liver in turn repackages cholesterol together with triglycerides into another lipoprotein called VLDL.
The function of VLDL is similar to chylomicrons, i.e. supply of free fatty acids to the muscle and adipose tissues and cholesterol to peripheral cells (Brown, M. S. and Goldstein, J. L Sci. American (1984)251, 58-66). In the circulatory system, triglycerides in the VLDL particle are hydrolyzed by an enzyme called lipoprotein lipase (LPL) and additional processing by hepatic lipase finally converts it to LDL (Dolphin, P. J.: Can. J. Biochem. Cell. Biol. (1985) 63, 850-869). Thus, the liver produces VLDL, the precursor of LDL. Because VLDL is a precursor to LDL, decreases in VLDL production translate into lowered LDL levels. High levels of circulating LDL have been positively correlated with the development of coronary disease. While LDL cholesterol is clearly an independent positive risk factor, HDL cholesterol is considered to be a negative risk factor (D. L. Tribble, R. M. Krauss. Advances in Internal Medicine. (1993) 38:1-29).
Apoprotein B
Apoprotein B-100 (apoB) is the major apoprotein of LDL and its ligand for the LDL receptor. ApoB-100 is a large protein with a molecular weight (MW) of 549,000. The protein is highly hydrophobic and is insoluble in the absence of lipids. The structure of apoB-100 has been studied using monoclonal antibodies raised to specific regions (antigenic regions or epitopes) on the protein. These monoclonal antibodies have been used to “map” the site on apoB-100, which binds to the LDL receptor. Mapping the various epitopes on apoB in VLDL and LDL using monoclonal antibodies has been a productive method to understand the role of various portions of this protein in lipoprotein uptake. Changes in the immunoreactivity of the epitopes on apoB to monoclonal antibodies have shown correlation with the uptake of LDL by cells in culture (N. F. Galeano, et al., J. Biol. Chem. vo. 269, no. 1, pp. 511-519, 1994).
Monoclonal antibodies have been used to bind to epitopes of a known region of apoB in order to determine the binding region for LDL receptor (Milne, R. et al., J. Biol. Chem. vol. 264, no. 33, 1988, pp. 10754-60; Milne, R. et al., Arteriosclerosis, vol. 3, no. 1, 1983, pp. 23-30). Assessment of the epitope position for the receptor blocking monoclonal antibodies can be used to predict the extent of the LDL receptor binding region of apoB (Pease, R., et al., J. Biol. Chem. vol. 266, no. 1, 1990, pp. 553-68).
Apolipoprotein E
Apolipoprotein E (apoE), like apoB, binds to the LDL receptors and is capable of transporting cholesterol throughout the system. ApoE mediates the transport and uptake of cholesterol and lipid by its high affinity binding with the LDL receptor. The LDL receptor recognizes both apoB and apoE with comparable affinity (Wilson, C., et al., Science vol. 252 pp. 1817-1822, 1991). The receptor binding domain of the apoE protein has been characterized via inhibition studies which utilized monoclonal antibodies (Weisgraber, K. H., et al., J. Biol. Chem., vol. 258, No. 20, Oct. 25, 1983, pp. 12348-12354). Further, the three-dimensional structure of the LDL receptor binding domain of apoE has been determined by x-ray crystallography (Wilson, C., et al., Science vol. 252 pp. 1817-1822, 1991).
Clearance of LDL from Circulation
LDL is removed from plasma by a high affinity receptor called the LDL receptor, present on the cell-surface of peripheral and liver cells (Goldstein, J. L. and Brown, M. S.: Ann. Rev. Biochem. (1977) 46, 897-930). This receptor-mediated pathway accounts for uptake and degradation of LDL by cells, and in the process, cholesterol is delivered to these cells. Thus uptake of LDL by the receptor mediated process permits cells to acquire cholesterol from the lipoprotein, and this in turn provides cholesterol for membrane synthesis in all tissues and steroid hormone synthesis in the adrenal, ovaries and testes.
Uptake by the LDL receptor pathway is the major mechanism of LDL clearance from the plasma. This process of lipoprotein uptake is a highly coordinated and orchestrated process dictated by apolipoprotein composition and lipid content of the lipoprotein. The apolipoprotein on LDL, called apoB-100, mediates the interaction of LDL with the LDL receptor. Specific amino acid sequences on apoB form the binding site for apoB to the cell-surface LDL receptors (Knott et al., Nature (1986) 323:734-738).
Besides LDL, VLDL can also bind to the LDL receptors since it contains apoB-100 as well as apoE, another apolipoprotein, which contains LDL-receptor recognizable amino acid sequences. It is well recognized that VLDL are heterogeneous with respect to size and composition, and each subclass of VLDL differ in their ability to interact with the LDL receptor. For example, the large sized VLDL do not normally bind to the LDL receptor even though they contain apoB-100 and apoE. Only smaller VLDL bind to the receptor. It is suggested that both apoB-100 and apoE in the large VLDL do not possess the appropriate three dimensional structure for receptor recognition. Similarly, certain types of LDL, for example those found in the diabetic plasma (glycosylated LDL) and oxidized LDL, also do not bind LDL receptors because of incorrect structure of apoB-100 (Wang X, Bucala R, Milne R. Proc. Natl. Acad. Sci (1998) 95:7643-7647). Thus besides the amino acid sequence requirement, there is also a strict structural requirement of apoB-100 for optimal LDL binding by the LDL receptor.
The significance of apoB and apoE receptors in LDL clearance is demonstrated by patients possessing a genetic predisposition to coronary disease. A condition known as familial hypercholesterolemia (FH) impairs the clearance of LDL from blood plasma in patients lacking apoB or apoE LDL-receptors or having defective apoB or apoE LDL-receptors (Innerarity, T., et al., Proc. Natl. Acad. Sci. USA, vol. 84, pp. 6919-23, 1987). At least one abnormal apoB species has been reported in humans. Young, et al. documented the existence of one such abnormal apoB protein, apoB-37 (J. Clin. Invest., Vol. 79, June 1987, 1831-1841). This abnormal protein was found to occur frequently in individuals suffering from a form of familial hypobetalipoproteinemia, with the abnormal protein coding alleles being inherited and traced through over three generations of an affected family.
Existing Lipid Lowering Therapies
Diet contributes up to 40% of cholesterol that enters through the intestine and bile contributes the rest of the “exogenous” cholesterol absorbed through the intestine (Wilson M. D., Rudel L. L.: J. Lipid Res. (1994) 35:943-955). Decreasing dietary cholesterol absorption therefore is a regulatory point for cholesterol whole body homeostasis. Cholesterol absorption inhibitors lower plasma cholesterol by reducing the absorption of dietary cholesterol in the gut or by acting as bile acid sequestrants (Stedronsky E R: Biochim. Biophys. Acta(1994) 1210:255-287).
Since it has been determined that hypercholesterolemia is due to elevated LDL (hyperlipidemia), the lowering of LDL levels by drug therapy is attempted. There are several drug classes that are commonly used to lower LDL levels, including bile acid sequestrants, nicotinic acid (niacin), and 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors. Probucol and the fibrate derivatives are sometimes used as adjunctive therapy, usually in combination with other medications.
Cholesterol lowering agents decrease total plasma and LDL cholesterol and may increase HDL. Neomycin, a non-absorbable aminoglycoside binds dietary cholesterol and prevents intestinal absorption. Another drug in this category is cholestyramine. Cholestyramine is an anion-exchange resin, which acts by binding bile acids within the intestinal lumen, thus interfering with their reabsorption and enhancing fecal excretion. Cholesterol-lowering agents in this class require large dosage and are usually associated with poor compliance and malabsorption of other nutrients and drugs.
Important classes of drugs that act on liver are fibrates. Fibrates are fibric acid derivatives (bezafibrate, fenofibrate, gemfibrozil, and clofibrate) which profoundly lower plasma triglyceride levels and elevate HDL (Sirtori C. R, Franceschini G.: Pharmac Ther. (1988) 37:167-191; and Grundy S. M., Vega G. L.: Amer J. Med. (1987) 83:9-20). The typical clinical use of fibrates is in patients with hypertriglyceridemia, low HDL and combined hyperlipidemia.
The mechanism of action of fibrates is not completely understood but involves the induction of certain apolipoproteins and enzymes involved in VLDL and HDL metabolism. Patient compliance with fibrates is good, but they are not drugs of choice for lowering LDL cholesterol. Nicotinic acid (niacin), a water-soluble vitamin has a lipid lowering profile similar to fibrates and may target the liver.
Statins represent a class of compounds which are inhibitors of HMG CoA reductase, a key enzyme in the cholesterol biosynthetic pathway (Endo A, In: Cellular Metabolism of the Arterial Wall and Central Nervous System. Selected Aspects. Schettler G, Greten H, Habenicht AJR(Eds.) Springer-Verlag, Heidelberg (1993)).
The statins decrease liver cholesterol biosynthesis, which increases the production of LDL receptors thereby decreasing plasma total and LDL cholesterol (Grundy S. M. New Engl. J. Med. (1988) 319:24-32 and Endo A: J. Lipid Res. (1992) 33: 1569-1582). Depending on the agent and the dose used, statins may decrease plasma triglyceride levels and may increase HDL. Currently the statins on the market are Lovastatin (Merck), Simvastatin (Merck), Pravastatin (Sankyo and Squibb) and Fluvastatin (Sandoz). A fifth statin, Atorvastatin (Parke-Davis/Pfizer), is the most recent entrant into the statin market. Statins have become the standard therapy for LDL cholesterol lowering. The statins are effective LDL lowering agents but have side effects, the most common being increases in serum enzymes (transaminases and creatinine kinase). In addition, these agents may also cause myopathy and rhabdomyolysis especially when combined with fibrates.
Another drug that in part may impact the liver is probucol (P. Zimetbaun, M.D., H. Eder, M.D., and W. Frishman, M.D.: Clin. Pharmacol. (1990) 30:3-9). Probucol is used primarily to lower serum cholesterol levels in hypercholesterolemic patients and is commonly administered in the form of tablets available under the trademark Lorelco™. Probucol is chemically related to the widely used food additives 2,[3]-tert-butyl-4-hydroxyanisole (BHA) and 2,6-di-tert-butyl-4-methyl phenol (BHT). Its full chemical name is 4,4′-(isopropylidenedithio) bis(2,6-di-tert-butylphenol). Probucol is a lipid soluble agent used in the treatment of hypercholesterolemia including familial hypercholesterolemia (FH). Probucol reduces LDL cholesterol typically by 10% to 20% but also reduces HDL by 20% to 30%. The drug has no effect on plasma triglycerides. The mechanism of action of probucol in lipid lowering is not completely understood. The LDL lowering effect of probucol may be due to decreased production of apoB containing lipoproteins and increased clearance of LDL. Probucol lowers LDL in the LDL-receptor deficient animal model (WHHL rabbits) as well as in FH populations. Probucol has been shown to actually slow the progression of atherosclerosis in LDL receptor-deficient rabbits as discussed in Carew et al. Proc. Natl. Acad. Sci. U.S.A. 84:7725-7729 (1987). The HDL lowering effect of probucol may be due to decreased synthesis of HDL apolipoproteins and increased clearance of this lipoprotein. High doses of probucol are required in clinical use.
U.S. Pat. No. 5,262,439 to Parthasarathy discloses analogs of probucol with increased water solubility in which one or both of the hydroxyl groups are replaced with ester groups that increase the water solubility of the compound. In one embodiment, the derivative is selected from the group consisting of a mono- or di-probucol ester of succinic acid, glutaric acid, adipic acid, seberic acid, sebacic acid, azelaic acid, or maleic acid. In another embodiment, the probucol derivative is a mono- or di-ester in which the ester contains an alkyl or alkenyl group that contains a functionality selected from the group consisting of a carboxylic acid group, amine group, salt of an amine group, amide groups, amide groups, and aldehyde groups.
WO 98/09773 filed by AtheroGenics, Inc. discloses that monoesters of probucol, and in particular, the monosuccinic acid ester of probucol, are effective in simultaneously reducing cholesterol, lowering LDL, and inhibiting the expression of VCAM-1, and thus these compounds are useful as composite cardiovascular agents. Since the compounds exhibits three important vascular protecting activities simultaneously, the patient can take one drug instead of multiple drugs to achieve the desired therapeutic effect.
WO 98/09781 discloses therapeutic agents for the treatment of diseases, including cardiovascular diseases, that are mediated by VCAM-1.
U.S. Pat. No. 5,807,884 claims a method for treating a disease (including a cardiovascular disease) mediated by VCAM-1 that includes administering a substance that inhibits the oxidation of a polyunsaturated fatty acid.
U.S. Pat. No. 5,811,449 covers the method for the treatment of cardiovascular disease that includes suppressing the expression of a redox-sensitive gene selected from the group consisting of MCP-1, IL-6 and thrombin receptor that includes administering an effective amount of a substance that prevents or minimizes the oxidation of a polyunsaturated fatty acid.
U.S. Pat. No. 5,846,959 claims a method for treating a cardiovascular disease mediated by VCAM-1 expression, that includes administering an effective amount of a substance which inhibits the oxidation of a polyunsaturated fatty acid in combination with another cardiovascular drug selected from the group consisting of lipid lowering agents, platelet aggregation inhibitors, antithrombotic agents, calcium channel blockers, angiotensin converting enzyme inhibitors, and β-blockers.
U.S. Pat. No. 5,750,351 claims a method to assess a test compound for its ability to treat a disorder mediated by VCAM-1 that includes evaluating the ability of the compound to inhibit the oxidation of a polyunsaturated fatty acid.
U.S. Pat. No. 5,773,209 claims a method for the prediction or assessment of redox-sensitive gene mediated disease in vivo, that includes quantifying the level of oxidized polyunsaturated fatty acid in the tissue or blood, or a mediator of inflammation that is induced by polyunsaturated fatty acid or an oxidized polyunsaturated fatty acid.
U.S. Pat. No. 5,773,231 claims a method for the evaluation of the sensitization of a host's vascular endothelial cells to polyunsaturated fatty acids or their oxidized counterparts, that includes challenging a host with a polyunsaturated fatty acid or oxidized polyunsaturated fatty acid and comparing to a population norm the resulting concentration of VCAM-1 or other mediator of inflammation expressed by a redox-sensitive gene on exposure to the polyunsaturated fatty acid or oxidized fatty acid.
U.S. Pat. Nos. 5,380,747 and 5,811,449 claim a method for the treatment of cardiovascular disease in humans that includes administering an effective amount of a dithiocarbamate of defined structure. U.S. Pat. No. 5,792,787 discloses a method for the suppression of VCAM-1 expression in humans that includes administering an effective amount of a dithiocarbamate of defined structure.
U.S. Pat. No. 5,877,203 directed to a method for treating inflammatory diseases by suppressing VCAM-1 expression in humans that includes administering a dithiocarbamate of defined structure.
A series of French patents disclose that certain probucol derivatives are hypocholesterolemic and hypolipemic agents: Fr 2168137 (bis 4-hydroxyphenylthioalkane esters); Fr 2140771 (tetralinyl phenoxy alkanoic esters of probucol); Fr 2140769 (benzofuryloxyalkanoic acid derivatives of probucol); Fr 2134810 (bis-(3-alkyl-5-t-alkyl-4-thiazole-5-carboxy)phenylthio)alkanes; FR 2133024 (bis-(4-nicoinoyloxyphenylhio)propanes; and Fr 2130975 (bis(4-(phenoxyalkanoyloxy)-phenylthio)alkanes).
U.S. Pat. No. 5,155,250 discloses that 2,6-dialkyl-4-silylphenols are antiatherosclerotic agents. The same compounds are disclosed as serum cholesterol lowering agents in PCT Publication No. WO 95/15760, published on Jun. 15, 1995. U.S. Pat. No. 5,608,095 discloses that alkylated-4-silyl-phenols inhibit the peroxidation of LDL, lower plasma cholesterol, and inhibit the expression of VCAM-1, and thus are useful in the treatment of atherosclerosis.
Since cardiovascular disease is the leading cause of death in North America, there is a need to provide new therapies for its treatment, especially those that work through a mechanism different from the current drugs and can be used in conjunction with them.
It is therefore an object of the present invention to provide a new method to lower plasma cholesterol, and in particular low density lipoproteins and very low density lipoproteins.
It is another an object of the present invention to provide an assay to assess the effectiveness of the new method to lower plasma cholesterol.